Paenibacillus Sp. Strain, Cultivation Method and Use of the Same

ABSTRACT

Provided are a new  Paenibacillus  sp. strain, the deposit number of which is CGMCC No. 8333, and a method for culturing the exopolysaccharide; and an exopolysaccharide having a structural formula as shown in formula (I) being produced by the strain, as well as a production method thereof and its use in promoting the propagation of  bifidobacterium . The exopolysaccharide shown by formula (I) has a moderate degree of polymerization (DP=15-30) and has the functions of promoting the propagation of  bifidobacterium , as well as adjusting human intestinal microflora.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of applicant Ser. No.15/109,133 filed on Jun. 30, 2016, which is the national phase entry ofInternational Application No. PCT/CN2014/091544, filed on Nov. 19, 2014,which is based upon and claims priority to Chinese patent applicationNo. CN201310752153.7 with the filing date of Dec. 31, 2013. The contentsof these related applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the field of microorganism, morespecifically, relates to a new Paenibacillus sp. strain, the cultivationmethod and the use of the same.

BACK GROUND

In early research, Paenibacillus was classified into Bacillus based onthe morphology. In 1994, by using PCR probe testing, Ash et al. analyzedthe 16S rRNA sequences for different strains of Bacillus, and found thatsome of the Bacillus are significantly different from other Bacillus interms of genotypic characteristics, and their 16S rRNA sequences arehighly specific. Therefore, Ash separated 11 strains such as Bacilluspolymyxin from Bacillus to form an independent genus, namelyPaenibacillus.

The type strain of Paenibacillus is Paenibacillus polmyxin ATCC 842^(T),wherein the cells are in the shape of rods, the optimal growthtemperature range is 28-30° C. and the main fatty acid is anteisosaturated fatty acid C15:0. The G+C content of Paenibacillus range is45-54 mol %. Generally, if the difference of G+C mol % between twostrains is more than 5%, the two strains could be determined asdifferent species (such determination can be made even if othercharacteristics are similar). The DNA homology analysis may finallydetermine the classification of the strains. It is also a way fordetermining new species. In optimum conditions, if the DNA homology ishigher than 70%, the strains belong to the same species. If the DNAhomology is higher than 20%, the strains belong to the same genus.

Nowadays, it is believed that the effects of the combination ofphenotype and DNA homology to classify species and genus are accurateand desirable.

Many microorganisms of Paenibacillus genus have the effects ofdisease-preventing and growth-promoting on plants. Therefore, they havea good usage potential in agricultural industry. The Paenibacillus mayproduce many kinds of bioactive substances such as enzymes, antibioticsubstances, phytohormones, flocculants, and etc. Most of these activesubstances are proteins, polypeptides and polysaccharides, and etc.

Microbial extracellular polysaccharides (EPS for short), in some extent,have been demonstrated with the functions of anti-hyperlipidemia,immunoregulatory and anti-tumor and etc. Therefore, it may serve as thefood additive. As the consumers are more and more concerned about thefood safety issues, how to obtain new food additives (for example,thickener, emulsifier, stabilizer, etc) with a clear source, stableyield, and diverse functions attracted more and more attentions fromresearchers.

Levan is a type of fructans constituted of fructose units linked with β(2→6) fructoside bonds in main chain and with or without a few thebranchs lingked by β (2→1) fructoside bonds. The levan with a lowpolymerization (DP for short) (DP=2-9) is usually calledfructooligosaccharide. The levan with a DP range of 10-30 is usuallycalled polyfructose. The levan with a DP higher than 40 is usuallycalled high-polyfructose. Some levans that originates from microorganismhave important biological activities such as anti-tumor, anti-virus,anti-hyperglycaemia, anti-hyperlipidemia and immunopotentiation, andthus they have a large usage potential in terms of medicines andfunctional foods.

There are three methods for producing levans in large quantitiesnowadays: the chemical synthesis method, the microbiologicalfermentation and enzymatic synthesis method. However, currently, thechemical synthesis method merely produces the trisaccharides formed byβ-glycosidic bonds. Although both plants and microorganisms may producelevans, currently the levans produced by microorganism fermentation areall of high molecular weights with high polymerization degrees, usually2×10⁶-100×10⁶ Da and the DP is far more than 40. However, nowadays theyield and saccharose conversion rate of manufacturing levans frommicrobiological fermentation are usually low. Also, other products suchas high polymers, glucoses, fructoses, and fructooligosaccharidescoexist in the fermentation broth. Thus, it is disadvantageous forlarge-scale purification of levans. On the other hand, the enzymaticsynthesis method for producing levans requires certain conditions suchas the particular pH, the temperature, and ect. to facilitate thereaction, which is complex and hard to control. Therefore, the strainwith high productivity of levans and high saccharose conversion rate isthe key for large-scale preparation of levans, especially for the levanswith moderate and low polymerization.

SUMMARY

The purpose of the present invention is to disclose a new Paenibacillussp. strain, the culture method for prepairing levan, and the use of thesame. The purpose of the present invention is realized by the followingtechnical solutions.

The first technical solution of the present invention is a Paenibacillussp. strain whose deposit number is CGMCC No. 8333.

The second technical solution of the present invention is the method forculturing Paenibacillus CGMCC No. 8333, which includes the followingsteps. The Paenibacillus CGMCC No. 8333 is inoculated onto the culturemedium for culturing under 15-40° C. with a pH value of 5.5-8.5. Thetemperature for culturing according to the present invention is 15-40°C., and preferably is 30° C.

The pH value for culturing according to the present invention is5.5-8.5, and preferably is 6.0.

The cultivation according to the present invention may be variousmethods for culturing microorganisms, such as liquid culturing, solidculturing and semi-solid culturing. It may be shaking culturing. It alsocan be submerged fermentation in the fermentation tank. The shakingculturing is preferable.

The inoculum dose of the culturing according to the present invention isconventional, preferably, 2%. The percentage is the volume percentage.

The culture medium according to the present invention is theconventional culture medium for Paenibacillus, preferably, TYC culturemedium.

The culturing according to the present invention is the conventionalculturing for Paenibacillus, and is preferably conducted under aerobicconditions.

The third technical solution of the present invention is the use ofPaenibacillus CGMCC No. 8333 in preparing extracellular polysaccharide.

The fourth technical solution of the present invention is theextracellular polysaccharide of Paenibacillus. The structural formula ofthe extracellular polysaccharide is shown as Formula (1):

wherein, n=15-30.

Preferably, the extracellular polysaccharide has an average molecularweight distribution of 2500-5000 Da; and/or has the appearance of purewhite filament or powder.

The extracellular polysaccharide is produced by conventionalextracellular-polysaccharide-producing Paenibacillus strains in theliteratures. Preferably, the extracellular polysaccharide is produced byPaenibacillus bovis sp. nov. BD3526 whose deposit number is CGMCC No.8333, and produced by mutant strains or derivatives obtained from theoriginal strain of Paenibacillus BD3526.

The Paenibacillus BD3526 with the Paenibacillus deposit number of CGMCCNo. 8333 has been deposited in China General Microbiological CultureCollection Center (CGMCC), and has been disclosed in Chinese patentapplication CN103740618A.

The fifth technical solution of the present invention is a method forpreparing the extracellular polysaccharide of the Paenibacillus,including the following steps:

(1) The Paenibacillus CGMCC No. 8333 is fermented to obtain afermentation broth;(2) The fermentation broth obtained in step (1) is heated for 10-30minutes under 95-100° C. Once cooled down to 15-25° C., the pH value isadjusted to 4.4-4.8. It stands for 3-5 hours. It is centrifuged toobtain the supernatant. 80-100% ethanol solution is added at a volume2-4 times as much as that of the supernatant. it stands overnight. It iscentrifuged to collect the precipitates. Percentages refer to the masspercentages of the ethanol solution;(3) The precipitates obtained in step (2) is dissolved in distilledwater with the temperature of 50-80° C. to obtain the solution ofprecipitates with the concentration of 0.5-1.0%. Percentages refer tothe mass-volume percentages of the solution of precipitates. Once thesolution is cooled down to 20-25° C., trichloroacetic acid is added intothe solution at a final percentage of 4%-10%. Percentages refer to themass-volume percentages of the solution. The mixture is stored at 4-10°C. overnights centrifuged to obtain the supernatant. The supernatant isdialyzed with a membrane with a molecular weight cut-off of 1000 Da toobtain the retentate which contains extracellular polysaccharides.(4) The the retentate obtained in step (3) is dried to obtain the crudeextracellular polysaccharides.

The step (1) is conducted by the Paenibacillus CGMCC No. 8333 to obtainthe fermentation broth. Wherein, the fermentation of step (1) is theconventional fermentation in the art. Preferably, the fermentation isthe fermentation for 72 hours under 30° C. Preferably, the fermentationis conducted in the liquid polysaccharide-producing culture medium. Theliquid polysaccharide-producing culture medium is composed of 10%saccharose, 1% casein tryptone, 0.5% yeast extract, 0.5% K₂HPO₄, 0.034%CaCl₂), and distilled water. Percentages refer to mass percentages ofthe liquid polysaccharide-producing culture medium. The inoculum dose ofthe fermentation is the conventional inoculum dose in the art,preferably, 1%. The percentage is the mass percentage of the liquidpolysaccharide-producing culture medium.

The step (2) is heating the fermentation broth obtained in step (1) for10-30 minutes under 95-100° C. Once cooled down to 15-25° C., the pHvalue is adjusted into 4.4-4.8. It stands for 3-5 hours. It iscentrifuged to obtain supernatant. 80-100% ethanol solution is addedwith a volume 2-4 times as much as that of the supernatant. It standsovernight. it is centrifuged to collect the precipitates. Percentagesrefer to the mass percentages of the ethanol solution. Wherein, thecentrifugal conditions in step (2) are conventional in the art,preferably, the centrifugation is conducted for 10 min at 14000 g.Preferably, the adjustment of pH value is adjusting the pH value to 4.6.Preferably, the ethanol solution in step (2) refers to a 95%-ethanolsolution, wherein the percentage refers to the mass percentage of theethanol solution. Preferably, the added volume of ethanol solution is 3times as much as that of the supernatant.

The step (3) is dissolving the precipitates obtained in step (2) indistilled water with the temperature of 50-80° C. to obtain the solutionof precipitates with the concentration of 0.5-1.0%. Percentages refer tothe mass-volume percentages of the solution of precipitates. Once thesolution is cooled down to 20-25° C., trichloroacetic acid is added intothe solution to a final percentage of 4%-10%. Percentages refer to themass-volume percentages of the solution. The mixture is stored at 4-10°C. overnights centrifuged to obtain the supernatant. The supernatant isdialyzed with a membrane with a molecular weight cut-off of 1000 Da toobtain the retentate which contains extracellular polysaccharides.Wherein, the dissolution in step (3) preferably is dissolving theprecipitates obtained in step (2) with distilled water with thetemperature of 60° C. to obtain the solution of precipitates whoseconcentration is 0.8%. The percentage refers to the mass-volumepercentage of the solution of precipitates. Once the solution is cooleddown to 25° C., trichloroacetic acid is added into the solution to reacha final percentage of 4% of trichloroacetic acid. The percentage refersto the mass-volume percentage of the solution. The step (4) is dryingthe retentate which contains extracellular polysaccharides obtained instep (3) to obtain the crude product of extracellular polysaccharides.Wherein, the drying in step (4) is the conventional drying in the art.Preferably, the drying is the vacuum freeze drying. More preferably, thedrying is a vacuum freeze drying for 72 hours under 0.160 mBar and −30°C. Preferably, the method for preparing the extracellular polysaccharideaccording to the present invention further includes the following steps:

(5) The crude product of extracellular polysaccharide obtained in step(4) is dissolved in 0.05 mol/L of Tris-HCl buffer with a pH value of7.60. The EPS solutions is chromatographed on a DEAE-Sepharose FFcolumn. The linear gradient elution is conducted with the Tris-HClbuffer and the Tris-HCl buffer which contains 0.2-1.2 mol/L-NaCl insequence. The flow rate is 2-6 mL/min. The eluent is monitored forcarbohydrate content with the sulfuric acid-phenol method in theliterature and The absorbance is measured at the wavelength of 490 nm.Based on tube numbers corresponding to the absorbance, the plot is drawnto obtain the elution curve A;(6) The dialyzed aqueous solution corresponding to the single peak inelution curve A obtained in step (5) is collected. It is dialyzed withdeionized water. The vacuum freeze drying is conducted to obtain acomponent B of extracellular polysaccharide;(7) The component B of extracellular polysaccharide obtained in step (6)is dissolved in the Tris-HCl buffer to prepare the solution. Thechromatography is conducted on DEAE-Sepharose CL-4B ion exchange column.The elution is conducted with the Tris-HCl buffer which contains 0.2-1.2mol/L-NaCl. The flow rate is 2-6 mL/min. The eluent is monitored forcarbohydrate content with the sulfuric acid-phenol method in theliterature and the absorbance is measured at the wavelength of 490 nm.Based on tube numbers corresponding to the absorbance, the plot is drawnto obtain the elution curve B;(8) The dialyzed aqueous solution corresponding to the single peak inelution curve B obtained in step (7) is collected. It is dialyzed withwater. It is dried.

In the present invention, preferably, 50-200 mg of the crude product ofextracellular polysaccharide obtained in step (5) is dissolved in 0.05mol/L of Tris-HCl buffer with a pH value of 7.60 to prepare the solutionwith a concentration of 5-20 mg/mL. The preferred flow rate in step (5)is 3 mL/min. The DEAE-Sepharose FF column in step (5) is conventional inthe art, preferrably, D1.6×100 cm.

The preferred flow rate in step (7) is 3 mL/min. The DEAE-SepharoseCL-4B ion exchange column in step (7) is conventional in the art,preferably, D1.6×100 cm.

The “overnight” according to the present invention is a conventionalterm in the art. The preferred duration for it is 4-24 hours.

The sixth technical solution of the present invention is the use of theextracellular polysaccharide whose structural formula is shown inFormula (1) in promoting the proliferation of bifidobacteria.

Wherein, the extracellular polysaccharide is produced by Paenibacillus,and preferably, by Paenibacillus bovis sp. nov. BD3526 whose depositnumber is CGMCC No. 8333. The extracellular polysaccharide is producedby the conventional method in the art, preferably, by the preparationmethod according to the present invention.

In the present invention preferably, the bifidobacteria is thebifidobacteria in intestinal flora. The intestinal flora may come fromhuman faecal samples, preferably, from faecal samples of 3-to-6-year-oldchildren. Preferably, the bifidobacteria is B. breve, B. longum, or B.infantis, and more preferably, B. breve. According to common sense inthe art, the preferred conditions mentioned above can be combineddiscretionarily to obtain preferred embodiments of the presentinvention.

All reagents and raw materials used in the present invention arecommercially available.

The positive effects and progress of the present invention lie in that:the present invention provides a new strain of Paenibacillus. Thetaxonomic status of this strain is Paenibacillus sp. It is desirable tobe named as Paenibacillus bovis sp. nov. in accordance with the namingmethod of International Committee Systematic Bacteriology (Gao et al.,2016, International Journal of Systematic and EvolutionaryMicrobiology). The discovery and utilization of the new strain enrichthe available microbiological resources, and make contributions forbetter use of Paenibacillus in future. The Paenibacillus BD3526according to the present invention can be used as a microbialtherapeutic agent and can also be used for preparing extracellularpolysaccharide. The present invention also provides an extracellularpolysaccharide of Paenibacillus with a single component and a moderatedegree of polymerization (DP=15-30). It can be obtained through thefermentation of Paenibacillus BD3526. It has similar effects to those ofthe commercialized fructooligosaccharide. Its preparation method is easyto conduct. It is conducive for purification. The inventors of thepresent invention have analyzed the physical & chemical properties andstructural composition of the extracellular polysaccharide throughrigorous experiments. The use for promoting the proliferation of B.infantis in vitro and adjusting the intestinal flora for adults in vitrois provided.

Deposit Information of Biological Materials

The Paenibacillus BD3526 of the present invention has been deposited inChina General Microbiological Culture Collection Center (CGMCC) sinceOct. 14, 2013, and the address for the deposition is Institute ofMicrobiology, Chinese Academy of Sciences, NO. 1-3 West Beichen Road,Chaoyang District, Beijing 100101, China. The deposit number for thestrain is CGMCC No. 8333. The systematic name of the strain isPacnibacillus SP., with the strain name of BD3526.

BRIEF DESCRIPTION OF THE DRAWINGS

Characteristics and beneficial effects of the present invention areillustrated below in combination of drawings.

FIG. 1 shows the colonial morphology of new Paenibacillus strain BD3526according to the present invention on TYC culture medium.

FIG. 2 shows the growth curve of Paenibacillus strain BD3526 accordingto the present invention. The horizontal axis refers to the time and thevertical axis refers to the OD value of the 100 μL-culture solution in96-well plate at 600 nm.

FIG. 3 shows the phylogenetic tree of 16S rRNA system of the newPaenibacillus strain BD3526 according to the present invention.

FIG. 4 shows the absorbance value OD at A490 nm and A280 nm of the crudeproduct of extracellular polysaccharide of the Paenibacillus strainBD3526 according to the present invention.

FIG. 5 shows the elution curve on DEAE-Sepharose ion exchange column forthe crude product of extracellular polysaccharide of the Paenibacillusstrain BD3526 according to the present invention; wherein, the verticalaxis refers to the absorbance value OD of the eluent through sulfuricacid-phenol colorimetric method at n=490 nm, and the horizontal axisrefers to the tube numbers.

FIG. 6 shows the elution curve on Sepharose CL-4B molecular sieve gelcolumn of the peak of charged part (F1) of the extracellularpolysaccharide of the Paenibacillus strain BD3526 according to thepresent invention; wherein, the vertical axis refers to the absorbancevalue OD of the eluent through sulfuric acid-phenol colorimetric methodwhere n=490 nm, and the horizontal axis refers to the tube numbers.

FIG. 7 shows the infrared spectrum of the purified extracellularpolysaccharide of the Paenibacillus strain BD3526 according to thepresent invention.

FIG. 8 shows the ¹³C-NMR spectrum of the purified extracellularpolysaccharide of the Paenibacillus strain BD3526 according to thepresent invention.

FIG. 9 shows the molecular weight determined by liquid chromatographyfor the purified extracellular polysaccharide of the Paenibacillusstrain BD3526 according to the present invention; wherein, the verticalaxis refers to intensity of the, refractive index reflecting the contentof polysaccharides in the elents and the horizontal axis refers to theretention time (min).

FIG. 10 shows the monosaccharide composition for the purifiedextracellular polysaccharide of the Paenibacillus strain BD3526according to the present invention; wherein, the vertical axis refers tothe intensity of refractive index caused by the existence ofmonosaccharide, and the horizontal axis refers to the retention time.(a) shows the diagram of fructose (reference), and (b) shows the acidichydrolysate of the purified extracellular polysaccharide of thePaenibacillus strain BD3526.

FIG. 11 shows the graph of the in-vitro proliferation of Bifidobacteriuminfantis facilitated by the purified extracellular polysaccharide of thePaenibacillus strain BD3526 according to the present invention. Wherein,the blank control group refers to the blank control (i.e. the relativebacterial flora content of the in-vitro culture solution of infant feceswithout polysaccharide added); the extracellular polysaccharide ofBD3526 refers to the relative increase value of the bacterial floracontent of the in-vitro culture solution of infant feces with 0.4% (w/v)of the extracellular polysaccharide of the Paenibacillus strain BD3526according to the present invention added; FOS refers to the relativeincrease in the bacterial flora content of the in-vitro culture solutionof infant feces with 0.4% (w/v) of the commercializedfructooligosaccharide added.

FIG. 12 shows the PCR-DGGE diagram of V3 area for total bacteria of thefecal cultures. Wherein, the blank control group refers to the sample offecal cultures with no polysaccharide added, FOS refers to the fecalcultures with the commercialized fructooligosaccharide added, and BD3526EPS refers to the fecal cultures with the purified extracellularpolysaccharide of the Paenibacillus strain BD3526 according to thepresent invention added.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is further illustrated by means of examples below.However, the present invention is not thus limited within the scope ofthe examples. For experimental methods without any indicated specificconditions, conventional methods and conditions may be selected.Otherwise, the selection can be made in accordance with the instructionsof the description of the commercial products. The room temperatureaccording to the present invention refers to the temperature ofoperating room for the experiment and is usually 25° C. The “overnight”according to the present invention is a conventional term in the art,and the preferred duration for it is 4-24 hours.

Paenibacillus hunanensis FeL05^(T) (ACCC 10718^(T)=CGMCC No. 1.8907^(T))and Paenibacillus polymyxa ATCC 842^(T) (CGMCC No. 1.4261^(T)) arepurchased from China General Microbiological Culture Collection Center(CGMCC).

Example 1 Acquisition of the New Microorganism According to the PresentInvention

1 ml yak milk samples Collected at Damxung County, Tibet AutonomousRegion, China is taken aseptically t and subjected to serial dilutionwith sterile normal saline. The diluent is spreaded onto the TYC agarevenly and then is cultured for 24-48 hours under 30° C. Severalsnot-like, thread-drawing single colonies are selected and transferredrespectively onto new TYC agar to obtain the purified colonies.

Example 2 Acquisition of the New Microorganism According to the PresentInvention and the Characteristics Thereof

Biolog microbial automatic detector (manufacturer: Biolog Inc.)identification assay: Biolog microbial automatic detector (manufacturer:Biolog Inc.) identification assay is based on the differences inmetabolism of individual carbon resource by different microorganisms. 95types of carbon sources or other chemical substances are selected andfixed together with the color developing agent onto the 96-well plate(A1 well is the negative control well). Bacterial suspension isinoculated and cultured for certain time. Characteristic fingerprint isgenerated by testing testing the turbidity caused by the growth ofmicroorganisms. The final identification results are obtained bycomparing the characteristic fingerprint with the standard strainprofiles database.

1) A plurality of single colonies according to the above Example 1 istaken and inoculated onto the liquid TYC respectively. They are culturedfor a 24 hours under 30° C.2) They are centrifuged for 20 min at the speed of 10000 r/min. Thesupernatant is discarded. 1 mL of sterilized saline is added. Themixture is shaken on a shaker for 5 min and centrifuged for 20 min atthe speed of 10000 r/min to remove the carbon sources therein. Thesupernatant is discarded. 1 mL of sterilized saline is added, and themixture is shaken on a shaker for 5 min.3) The suspension is further diluted with sterile saline (NaCl, 0.85%)to make a suspension of adsorbance at 590 nm at 0.13±0.02.4) The above suspension is added onto the Biolog ECO microplate (150μL/well). It is cultured under 20° C. The Biolog bacteria automaticreadout device reads the data every 12 hours for 2 days continuously.The results are shown in Table 1.

TABLE 1 BIOLOG IDENTIFICATION RESULTS FOR STRAIN BD3526. PROB SIM DISTspecies 1 0.535 0.535 6.927 Virgibacillus sediminis 2 0.117 0.117 7.531Brachybacterium paraconglomeratum 3 0.102 0.102 7.716 Peanibacillustundrae 4 0.095 0.095 7.802 Peanibacillus polymyxa

Three parameters should be taken into consideration to identify theresults: Probabilities (PROB), Similarities (SIM), and Distances (DIS).SIM and DIS values are two important parameters, which indicates thematching degrees between the tested results and the corresponding dataof the database. When DIS<5.0 and SIM>0.75, the matching is good. Theresults show that the identified SIM value of the strain BD3526 is0.535<0.75, indicating a low matching degree with the data of thedatabase. This shows that it has a significant difference from thestrains of the database in terms of metabolic characteristic. Itprobably is a new species of microorganism.

The strain BD3526 has been deposited in China General MicrobiologicalCulture Collection Center (CGMCC) since Oct. 14, 2013, and the addressfor the deposition is: Institute of Microbiology, Chinese Academy ofSciences, NO. 1-3 West Beichen Road, Chaoyang District, Beijing 100101,China. The deposit number for the strain is: CGMCC No. 8333. Thesystematic name of the strain is Paenibacillus SP., with the name ofBD3526.

Example 3 Characteristics of the New Microorganism According to thePresent Invention 1. Colony Characteristics:

The single colonies of strain BD3526 is taken and transferred onto theTYC agar (agar). They are cultured aerobically at 30° C. for 24 hours,36 h, and 48 h. The characteristics of colonies such as size, color,edge, embossment, smoothness, viscidity, and transparency are observedrespectively. The results are shown in FIG. 1. The results show that onTYC agar, the strain BD3526 forms irregularly-edged, smooth, ropy,glossy-surfaced, and non-transparent colonies. The diameters are of 3-5mm.

2. Morphological Observation and Physiological and BiochemicalCharacteristics:

The fresh cultures which have been cultured on TYC solid culture medium(agar) for 24 hours are picked up for physiological and biochemicaltests. The results show that BD3526 is Gram-positive bacillus withterminal spore, in the shape of an oval, and not expanded. Theparameters for physical and chemical reactions of BD3526 are shown inTable 2.

TABLE 2 results for physical and chemical tests on strain BD3526Oxidase− Catalase+ β-galactosidase+ arginine double Lysine Ornithinehydrolysis of− decarboxylase− decarboxylase+ Urease− Citrateutilization− Nitrate reduction+ Indole production− VP reaction+ H₂Sproduction− Amylolysis+ Esculin hydrolysis+ Gelatin liquefaction+

3. API 50 CHB Identification Characteristics

The acid produced from fermentable carbohydrates by strain BD3526 isdetermined using API 50 CHB identification system (manufacturer: bioMe′rieux). In the reagent strips of API 50 CHB, different serial numberscorrespond to different carbon sources. Meanwhile, the indicator iscontained therein. Thus, if the corresponding carbon source ismetabolized and acids are produced from the corresponding substrate, thepH value of the culture solution will decrease, and the color of theindicator will change. It is easy to observe and record.

1) The components of API 50 CHB basic culture medium are: 1 g oftryptone, 0.5 g of yeast extract, 2 g of ammonium sulfate, 0.18 g ofphenol red, 10 ml of the inorganic salt base (Cohen-Bazire), and 1000 mlof phosphate buffer (pH7.8).2) Freshly cultured colonies of BD3526 on TYC agar are picked andsuspended into the sterilized saline, and transferred to the culturemedium according to 1) to prepare a bacterial suspension withOD₆₀₀=0.4-0.6.3) The bacterial suspension in 2) is then inoculated the the small tubeson API 50 CHB test strips. A layer of sterilized paraffin oil is addedinto the tubes to cover the surface.4) The inoculated tubes are cultured at 30° C. The change of color ofthe medium in the tubes (originally blue-purple, in the case of acidproduced, the color would changed to yellow) are recordered at 24 and 48hours respectively.

The results for acid produced from fermented carbohydrates by strainBD3526 are shown in Tables 3 and 4.

TABLE 3 API 50 CHB identification results of strain BD3526 Control−D-galactose+ Glycerol− Melibiose+ Gentiobiose+ Mannitol+ D-glucosee+Maltose+ Saccharose+ D-lyxose− Erythritol− D-fructose+N-acetyl-glucosamine+ Xylitol− D-tagatose− D-arabinose− D-mannose+Amygdalin+ Raffinose+ D-fucose− L-arabinose+ L-sorbose− Arbutin+Melezitose− L-fucose− D-ribose+ L-rhamnose− Esculin+ Lactose+D-arabitol− D-xylose+ Dulcitol− Salicin+ Starch+ L-arabitol− L-xylose−Iinositol− Cellobiose+ Glycogen+ Gluconate+ Adonitol−α-methyl-D-mannoside− α-methyl-glucoside− 5-keto-gluconate−2-keto-gluconate+ β-methyl-D-xylose+ Sorbitol− “+” refers to acidproduced from sugar, and “−” refers to no acid produced.

TABLE 4 The acid producing profiles from metabolizable carbohydrates bystrain BD3526 and similar Paenibacillus sp. strain Strains 1 2 Glycerol− w Iinositol − w Gluconate + w α-methyl-glucoside − w2-keto-gluconate + − Trehalose w + Note: strain 1 refers to BD3526, andstrain 2 refers to Paenibacillus hunanensis FEL05^(T) (ACCC 10718¹); WREFERS TO “WEAK”, i.e., WEAK POSITIVE.

As shown in Table 4, the strain BD3526 and Paenibacillus hunanensisFeL05^(T) (ACCC 10718¹) displayed different ability in terms of acidproduction from metabolizable carbohydrates, indicating that the twostrains belong to different species.

Example 4 Growth Characteristics of the New Microorganism According tothe Present Invention 1. Growth Curve:

1) 30 mL of TYC liquid culture medium is added to the 100 mL erlenmeyerflasks respectively, and sterilized at 121° C. for 15 minutes;2) The freshly cultured colonies of strain BD3526 on TYC solid culturemedium (agar) for 24 hours are inoculated into the TYC liquid culturemedium as mentioned above, and incubated on a shaker at 30° C. for 20-24hours to obtain the inocula.3) The inocula of BD3526 obtained in 2) are transferred to the fresh TYCliquid culture at a ratio of 2% (v/v) and thoroughly mixed. The mixtureis added into the wells on a costar 96-well sterile microwell plate, 150μl for each well, and the experiment is taken out in triplicates,employing non-inoculated TYC liquid culture medium as the control. Theadsorbance of the wells at 600 nm are recorded at an interval of 30 min.The results are shown in FIG. 2.

2. Growth Temperature:

The inocula of BD3526 obtained in 2) are transferred to tubes containing5 mL of the fresh TYC liquid culture at a ratio of 2% (v/v) andthoroughly mixed. The inoculated tubes are cultured in Water bath at 4°C., 15° C., 30° C., 37° C., 40° C. and 60° C., respectively, intriplicates for each temperature gradient. The turbidity of the tubesare recorded at 24 hours and 48 h respectively to determine the growthof the strain BD3526, empolying non-inoculated TYC liquid culture mediumas the control. The obtained growth temperature range of strain BD3526is 15-40° C., preferably, 30° C.

3. NaCl Tolerance for Growth

The inocula of BD3526 obtained in 2) are transferred to at a ratio of 2%(v/v) to tubes containing 5 mL of the fresh TYC liquid culture with asodium chloride concentration of 0.0%, 2.0%, 5.0%, 7.0% and 10.0%respectively and cultured at 30° C. The turbidity of the tubes arerecorded at 24 hours and 48 h respectively to determine the growth ofthe strain BD3526, empolying non-inoculated TYC liquid culture medium asthe control. The results show that the NaCl tolerance for strain BD3526is 10%.

4. pH Range for Growth

The pH value of the sterile TYC culture medium is adjusted to 3.0, 4.0,5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5, 9.0 and 10.0 using sterile HCl andNaOH. The inocula of BD3526 obtained in 2) are transferred to at a ratioof 2% (v/v) to tubes containing 5 mL of the fresh TYC liquid culture ofindividual pH value and cultured at 30° C. The turbidity of the tubesare recorded at 24 hours and 48 h respectively to determine the growthof the strain BD3526, empolying non-inoculated TYC liquid culture mediumwith individual pH value as the control. The obtained pH range forgrowth of strain BD3526 is 5.5-8.5, and preferably is 6.0.

Example 5 the 16S Phylogenetic Characteristics for the New MicroorganismAccording to the Present Invention

The genomic DNA of strain BD3526 is obtained in accordance with theoperation procedure for Gram-positive bacteria using TIANAMP BacteriaDNA Kit. The absorbance thereof is determined at 230 nm, 260 nm, and 280nm. The A260: A280: A230 thereof is 1:0.510:0.445. The purity meets therequirements.

The fragment of 16S rDNA of strain BD3526 is amplified using 27F,1492R-primer. The amplified fragments are purified and then ligated tothe TA cloning vector of pMD19-T Simple Vector. Then, they are put intothe water bath of 16° C. overnight. They are transformed into thecompetent cell of E. coli DH5a. They are spread onto the LB agar culturemedium plate with ampicillin. They are culturied under 37° C. for 16-20hours. The positive transformants are picked up. The positivetransformants mentioned above are sent to JIE LI BIOLOGY Co. in Shanghaifor sequencing. The results for sequencing are put into the database ofNCBI and EzTaxon, and the most similar strain found through comparing isPaenibacillus hunanensis FeL05^(T) with a similarity of 96.6%.

Sequences for the primer pair mentioned above are: for 1492R:TACCTTGTTACGACTT, and for 27F: AGAGTTTGATCCTGGCTCAG.

The result of gene sequencing of 16S rRNA of strain BD3526 is shown inSEQ ID NO. 1.

The sequence of 16S rRNA mentioned above is compared by using thesoftware of CLUSTAL_X program (version 1.83) and the phylogenic tree isdrawn by MEGA version 4.0.2. software. Using neiglibor-joining forcalculation with maximum-parsimony and maximum-likelihood forverification calculation, the bootstrap is set as 1000 cycles. Theresults are shown in FIG. 3. As shown in FIG. 3, through the analysis onphylogenetic tree of 16S rRNA gene, strain BD3526 shall be classified asthe cluster of Paenibacillus hunanensis. However, the similarity betweenstrain BD3526 and the type strain of Paenibacillus hunanensis is 96.6%.However, similarity of 16S rRNA gene lower than 97% is the threshold todescriminate different species strains in the same genus. Moreover, muchevidence has been proven that for some bacteria with the similarity over99% on gene sequence for 16S rRNA are still classified as differentspecies. Thus, the strain BD3526 is likely to be a new species ofmicroorganism with its other physiological and biochemical indexes to beverified.

Example 6 Characteristics on Fatty Acid Content for the NewMicroorganism According to the Present Invention

The determination of total fatty acid content of the strain BD3526.

The following solutions are prepared: I, 45 g of sodium hydroxide isdissolved in 150 ml of methanol and 150 ml of distilled water; II, 190ml of concentrated hydrochloric acid and 275 ml of methanol aredissolved in 135 ml of distilled water; III, 200 ml of normal hexane ismixed with 200 ml of ethyl ether homogeneously; IV, 10.8 g of sodiumhydroxide is dissolved in 900 ml of distilled water; and V, saturatedsodium chloride solution.

1) A certain amount of the bacterial culture is taken and added into a 8ml-screwed glass tube. 1 ml of solution I is added. The screw cap of thetube is fastened and put into the boiling water bath for 5 min. It istaken out and shaken for 5 to 10 seconds. It is put into the boilingwater bath for 25 min.2) Once the sample tube is cooled down, 2 ml of solution II is addedinto it. The tube is capped, shaken. and bathed for 10 min at 80±1° C.with an accurate control. Then, it is cooled down with ice bath.3) 1.25 ml of solution III is added into the solution mentioned above.It is rapidly shaken for about 10 min. The lower aqueous phase isdiscarded.4) 3 ml of solution IV and 0.1-0.2 ml of solution V are added into theremaining organic phase. It is rapidly shaken for about 5 min. Twothirds of the upper organic phase is taken and put into the samplebottle of chromatography.

The HP6890 gas chromatograph is equipped with split/splitless inlet,hydrogenation flame ionization detector (FID) and HP gas chromatographchemstation; the chromatographic column is Ultra-2 column with thelength of 25 m, the inner diameter of 0.2 mm, and the liquid filmthickness of 0.33 μm. The furnace temperature is two step programmedrising temperature. The initial temperature is 170° C. The temperatureis raised up to 260° C. in the rate of 5° C./min and then raised up to310° C. in the rate of 40° C./min and maintained for 1.5 min. Thetemperature at inlet is 250° C. and the carrier gas is hydrogen with theflow rate of 0.5 ml/min in split mode. The split ratio is 100:1. Thesample size is 2 μl. The temperature for test is 300° C., the flow rateof hydrogen is 30 ml/min, the flow rate of air is 216 ml/min and theflow rate of supplemental gas (nitrogen) is 30 ml/min.

The results show that the main cellular fatty acids of strain BD3526 areanteiso saturated fatty acid C_(15:0), anteiso heptadecenoic saturatedfatty acid, and hexadecanoyl saturated fatty acid. The percentages ofthe contents are 59.02%, 11.09%, and 7.66% respectively. The main fattyacid in accordance with the Paenibacillus is anteiso saturated fattyacid C_(15:0). Both the type and the content of the fatty acid thereofare different from those of the similar strains. Therefore, the strainis determined as a different species from similar strains.

Example 7 Characteristics on G+C Mol % Content for the New MicroorganismAccording to the Present Invention

The determination for G+C mol % content of genomic DNA for strainBD3526.

The melting temperature (Tm) method is used. E. coli K12, AS 1.365 isused as the reference control. The device used is the Lambda35 UV/VISSpectrometer of Perkin/Elmer. The temperature is controled by PTP-1temperature digital controller. The steps are as follows:

1) The sample DNA to be tested is diluted with 0.1×SSC such that itsOD_(260 mm) value ranges from 0.3 to 0.4;2) The OD value is recorded under 25° C. at the wavelength of 260 nmfirstly. Then the temperature raising procedures is set up to raise thetemperature from 65° C. to 95° C. at the rate of rising 1° C. perminute;3) The raising of OD value indicates the beginning of denaturation. Thetemperature of cuvette and the OD value are recorded until the OD valueremains unchanging, which indicates the completion of denaturation; and4) The melting temperature (Tm) is obtained based on the thermaldenaturation curve and calculating the G+C mol % content.

The calculation formula in 0.1×SSC solution is:

G+C mol %=G+C mol %AS _(1.365)+2.08(Tm _(unknown)

Tm _(AS1.365))

The Tm of E. coli K12, AS_(1.365) determined in the test is 75.810° C.The Tm value and G+C mol % of the strain to be tested.

The results of G+C mol % of strain BD3526 are shown in Table 5.

TABLE 5 the G + C mol % content for strain BD3526 Strain number Tmvalue, ° C. G + C mol % Control E coli kl2 75.810 — BD3526 74.024 47.48

The G+C mol % of strain BD3526 is 47.48%, The G+C mol % of Paenibacillushuncmensis FeL05^(T) (ACCC 10718^(t)=CGMCC 1.8907^(T)=DSM22170^(T)) G+Cmol % is 53.3%. The difference between the two strains is greater than5%. The G+C content of Paenibacillus range is within 45-54 mol %.According to the “Common Bacteria Identification System Manual”(Dongfang Xiu, Cai Miaoying), the G+C content of Paenibacillus range iswithin 45-54 mol %; the difference of G+C mol % between the two strainsis greater than 5%. Therefore, these two strains can be determined asdifferent species (such determination can be made even if othercharacteristics are similar). Therefore, the strain BD3526 is classifiedas Paenibacillus sp. which belongs to a different species with respectto its most-similar strain Paenibacillus hunanensis FeL05^(T) (ACCC10718^(T)=CGMCC 1.8907^(T)=DSM 22170^(T)).

Example 8 Hybridization Experiment for the New Microorganism Accordingto the Present Invention

The hybridization experiment between strain BD3526 and the strain withthe most related genetic relationship, and the hybridization experimentbetween strain BD3526 and the type strain of Paenibacillus.

Referring to the results of 16S rRNA, DNA-DNA hybridization experimentbetween strain BD3526 and the species of Pamibacillus hunanensis FeL05^(T) (ACCC 10718^(T)=CGMCC 1.8907^(T)=DSM 22170^(T)) with the mostrelated genetic relationship is conducted, and DNA-DNA hybridizationexperiment between strain BD3526 and the type strain Pamibacilluspolymyxa ATCC 842^(T) (=CGMCC 1.4261^(T)=DSM 36^(T)=KCTC 3858^(T)) ofPamibacillus is conducted.

The method of liquid phase renaturation rate is used. The device used isthe Perkin Elmer Lambda35 UV/VIS Spectrophotometer. The temperature iscontrolled by PTP-1 Peltier System digital temperature controllingsystem. The steps are as follows.

1) DNA sample processing: the DNA sample is extracted as described inExample 5 above. Before the experiment, it is ice-bathed and zapped withthe ultrasonic wave of 40 W for 24 minutes (the setting is: zapping for3 seconds/pausing for 3 seconds). The concentration of DNA sample isOD₂₆₀ nm of 2.0. The DNA sample is cut into fragments of 2-5×10⁵daltons.2) The DNA samples (A, B) to be tested are precisely prepared using0.1×SSC respectively into a status where the OD₂₆₀ nm value range is1.8-2.0. The OD₂₆₀ nm value for both of them shall be consistent (asaccurate as 0.001).3) After entering the UV Winlab program (manufacturer: Perkin Elmer),the method window appears. The “Time-Driven” (TD) method in the methodwindow is selected. Proper parameters are set on the “Timed, Inst.Sample.” setting page. The wavelength for determination is 260 nm andthe total time for determination is set as 30 minutes. The optimalrenaturation temperature (TOR) on the basis of the G+C mol %. Thetemperature of cuvette is kept stable at the optimal renaturationtemperature. In 2×SSC reaction liquid, the optimal renaturationtemperature is calculated by the formula: TOR=0.51×(G+C) mol %+47.4) 400 μl of the DNA samples of each of the two strains is added intotwo centrifugal tubes separately. 200 μl of the DNA samples of each ofthe two strains is added into the same centrifugal tube to form themixed sample.5) Before testing, the single DNA sample and mixed DNA sample should bedegenerated for 15 min under 100° C. through PTP-1 temperature-controlsystem (manufacturer: Perkin Elmer), respectively. They are cooled downto the optimal renaturation temperature. The OD₂₆₀ nm value is recorded.The reading does not stop until 30-min of the reaction. The temperatureof samples should not be lower than TOR during the whole process.Finally, a straight line showing a gradually decreased absorbance valuewith time is be obtained.6) Based on the software UV Winlab, select “Slope” in “Algorithm” columntherein to obtain the renaturation rate (V), namely, slope (“V” usuallyrepresents the reduced amount of absorbance per minute).7) The homogenous hybridization rate is calculated by the formula.

Homogenous hybridization rate(H)%=4Vm−(Va+Vb)/2√{square root over(VaVb)}×100%

The results of DNA-DNA hybridization is as follows:

BD3526/Paenibacillus hunanensis FeL05^(T) (repeat for three times):

H %=39.82% (I); H %=41.60% (II); and H %=42.10% (III).

BD3526/Paenibacillus polymyxa ATCC 842^(T) (repeat for three times):

H %=41.62% (I); H %=46.60% (II); and H %=48.60% (III).

The results show that the DNA homology between the strains BD3526 andPaenibacillus hunanensis FeL05^(T) (ACCC 10718^(T)=CGMCC 1.8907¹=DSM22170¹) is 39.82˜42.10%. With respect to the type strain Paenibacilluspolymyxa ATCC 842^(T) (=CGMCC 1.4261^(T)=DSM 36^(T)=KCTC 3858^(T)) ofPaenibacillus, the DNA homology is 41.62-48.60%. Based on “Bergey'sManual of Determinative Bacteriology”, under the optimum conditions, ifthe DNA homology is greater than 70%, they belong to the same species;and if the DNA homology is greater than 20%, they belong to the samegenus. Together with the data of Examples 2, 3, 4, 5 and 6, it isdetermined that the strain BD3526 belong to a new species ofPaenibacillus. The taxonomic status of this strain is Paenibacillus sp.It is likely to be named as Paenibacillus damxungensis sp. nov. inaccordance with the naming method of International Committee SystematicBacteriology. Moreover, the strain BD3526 is selected as the type strainof this species.

Example 9 Use of the New Microorganism According to the PresentInvention

As shown in FIG. 1, the colonies of strain BD3526 are viscous,indicating a high yield of extracellular polysaccharide thereof. Thesingle colonies mentioned above are picked up and inoculated onto theTYC liquid culture medium for fermentation. The products are obtainedafter ethanol precipitation. They are identified as polysaccharides. Thepolysaccharide is produced by the natural fermentation of strain BD3526.It is safe, nontoxic, and may serve as an emulsifier, thickener,stabilizer, gelatinizer, and etc. for industrial applications. StrainBD3526 can also be used directly in the form of a microbial therapeuticagent.

Example 10 Preparation for Extracellular Polysaccharide of the StrainBD3526 (1) The Fermentation of Paenibacillus and the Preparation forFermentation Broth

10.0 mg of the freeze-dried powder of Paenibacillus BD3526 is dissolvedwith 1 mL of sterile distilled water (100 mg/0.1 ml, impossible!). Oneloop of the suspension is picked and streaked on the solidpolysaccharide-producing culture medium (the solidpolysaccharide-producing culture medium is consisted of 1.2% of agar,10% of saccharose, 1% of casein tryptone, 5% of yeast extract, 5% ofK₂HPO₄, 0.034% of CaCl₂), and distilled water, wherein the percentagesrefer to the mass percentages with respect to thesolid-polysaccharide-producing culture medium). It is aerobicallycultured for 24 hours at 30° C.

The single colony formed on the solid polysaccharide-producing culturemedium is picked up and transferred into the liquidpolysaccharide-producing culture medium (consisting of 10% ofsaccharose, 1% of casein tryptone, 0.5% of yeast extract, 0.5% ofK₂HPO₄, 0.034% of CaCl₂), and distilled water, wherein the percentagesrefer to the mass percentages with respect to the liquidpolysaccharide-producing culture medium). It is cultured for 24 hoursunder 30° C. to obtain the inocula. The inocula is transferred intofresh liquid polysaccharide-producing culture medium at a ratio of 2%(v/v). It is cultured for 72 hours under 30° C. to obtain thefermentation broth.

(2) The Preparation of the Crude Product of Extracellular Polysaccharidein the Fermentation Broth

(2.1) The fermentation broth obtained in step (1) is heated under 100°C. for 10 minutes. It is cooled down to 25° C. The pH value is adjustedto 4.60 with food-grade lactic acid or hydrochloric acid. It stands for4 hours. It is centrifuged for 10 min (14000 g). The supernatant istaken. 3 volumes of 95%-ethanol solution to that of the supernatant areadded under gentle stirring and the mixture stands overnight. It iscentrifuged for 10 min (14000 g). The precipitates are collected. Thepercentage refers to the mass percentage of the ethanol with respect tothe ethanol solution.(2.2) The precipitate obtained in step (2.1) is dissolved in distilledwater at 60° C. to reach a final concentration of 0.8%. The percentagerefers to the mass-volume percentage of the solution of precipitates.When the solution is cooled down to 25° C., trichloroacetic acid isadded into the solution to a final concentration of 4% (w/v). Thesolution stands for 16 hours under 4° C. It is centrifuged or filteredto remove precipitates. The supernatant is dialyzed using a dialysis bag(spectrumlabs, U.S.) with a molecular weight cut-off of 1000 Da againstdistilled water for 72 hours. Water is changed every 12 hours. Theretentate is obtained.(2.3) The retentate obtained in step (2.2) is vacuum freeze dried underthe condition of 0.160 mBar, −30° C. for 72 hours to obtain the crudeproduct of extracellular polysaccharide. The purity of the crudeextracellular polysaccharide is checked by determining its absorbance at490 nm via sulfuric-phenol method or directly at 280 nm. The results areshown in FIG. 4. A490 nm is the characteristic peak of polysaccharide.A280 nm is the characteristic peak of protein. The results show thatthere is only one symmetric peak at 490 nm, indicating that the contentof crude proteins in extracellular polysaccharide is ignorable.Meanwhile, based on the calibration curve of standards (glucose orbovine serum albamin respectively) at A490 nm and A280 nm, it is knownthat the crude product of extracellular polysaccharide has a highpurity. The extracellular polysaccharide content is 95.7%, Thepercentage refers to the mass percentage of the crude product ofextracellular polysaccharide.

Example 11 Purification for the Crude Product of ExtracellularPolysaccharide

(1) 100 mg of the crude product of the extracellular polysaccharideobtained in Example 10 is dissolved in Tris-HCl buffer (0.05 mol/L, pH7.60) to prepare the solution with a concentration of 10.0 mg/mL. Thechromatography is performed on the DEAE-Sepharose FF column (D1.6×100cm) (GE Healthcare). The linear gradient elution is conducted with theTris-HCl buffer and in turn the Tris-HCl buffer (0.05 mol/L, pH 7.60)which contains 0.2-1.2 mol/L-NaCl. The flow rate is 3 mL/min. The liquidis collected in tubes (6 mL per tube). The sulfuric acid-phenol method(Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith,1956. Colorimeteric method for determination of sugars and relatedsubstances. Anal. Chem. 28:350-356) is used for tracking and monitoring.The absorbance is determined at the wavelength of 490 nm (i.e. thecharacteristic wavelength for the polysaccharide). Based on tube numberscorresponding to the absorbance, the plot is drawn to obtain the elutioncurve A. The data of the elution curve are shown in Table 6. Thus, theprimarily purified component of polysaccharide is obtained. That is,peak Fl (refer to FIG. 5) may comprise two or more differentpolysaccharide components. Further purification for verification isrequired.

TABLE 6 data of elution curve A Tube number OD₄₉₀NM 2 0.0711 6 0.0597 100.0539 14 0.0758 18 0.062 22 0.06 26 0.063 28 0.083 29 0.093 30 0.112 310.1845 32 0.3545 33 0.7526 34 1.0895 35 1.1524 36 1.0752 37 0.8605 380.4123 39 0.2715 40 0.1628 41 0.098 42 0.085 43 0.078 47 0.068 51 0.0752 0.0656 55 0.0731 58 0.0664 63 0.0671 67 0.0723 71 0.0689 75 0.0742 790.0853 81 0.0725 82 0.0834 83 0.0923 84 0.0808 85 0.0946 86 0.0989 870.0873 88 0.0622 89 0.0676 90 0.0648(2) The aqueous solution obtained through the elution from the tube withthe number corresponding to the single peak (F1) obtained in FIG. 5 instep (1) is collected. It is dialyzed with a 1000 Da-dialysis bag indeionized water for 72 hours. The vacuum freeze drying (0.160 mBar, −30°C.) is conducted for 72 hours to obtain the component B of extracellularpolysaccharide.(3) The component B of extracellular polysaccharide obtained in step (2)is dissolved in the Tris-HCl buffer to prepare the solution with theconcentration of 10.0 mg/mL. The chromatography is performed on the ionexchange column (the packing is DEAE-Sepharose CL-4B; column: D1.6×100cm, GE Healthcare). The elution is conducted using the Tris-HCl bufferwhich contains 0.2-1.2 mol/L NaCl. The flow rate is 3 mL/min. The eluentis collected in tubes (6 mL per tube). The sulfuric acid-phenol methodis used for tracking and monitoring. The absorbance at the wavelength of490 nm is determined. Based on the tube numbers corresponding to theabsorbance, the plot is drawn to obtain the elution curve B. Theexperimental results are shown in FIG. 6. There is only one single peakF2 in FIG. 6, indicating that only one component of polysaccharide iscontained. Moreover, F2 is the component of polysaccharide obtainedthrough the further purification of the primarily purified component F1of polysaccharide.(4) The aqueous solution obtained through chromatography from the tubeswith numbers corresponding to the single peak (F2) in FIG. 6 in step (3)is collected. It is dialyzed with a 1000-Da-dialysis bag in deionizedwater for 72 hours. The vacuum freeze drying is conducted under thecondition of 0.160 mBar, −30° C. for 72 hours to obtain the purifiedextracellular polysaccharide, wherein the polysaccharide content is 100%with no proteins contained. The percentage refers to the mass percentageof the extracellular polysaccharide.

The purified extracellular polysaccharide prepared in Example 10 and 11will be used in examples 14 to 18.

Example 12 Preparation for Extracellular Polysaccharide of the StrainBD3526 I Preparation for Polysaccharide of Strain BD3526 (1) TheFermentation of Paenibacillus and the Preparation for Fermentation Broth

10.0 mg of the freeze-dried powder of Paenibacillus BD3526 is dissolvedin 0.1 mL of sterile distilled water. The strain is picked up with aninoculating loop and streaked on the solid polysaccharide-producingculture medium (consisting of 1.2% of agar, 10% of saccharose, 1% ofcasein tryptone, 0.5% of yeast extract, 0.5% of K₂HPO₄, 0.034% ofCaCl₂), and distilled water, wherein the percentages refer to the masspercentages with respect to the solid polysaccharide-producing culturemedium) and cultured aerobically at 30° C. for 24 hours. The singlecolonies are picked up from the solid polysaccharide-producing culturemedium and transferred into the liquid polysaccharide-producing culturemedium (consisting of 10% of saccharose, 1% of casein tryptone, 0.5% ofyeast extract, 0.5% of K₂HPO₄, 0.034% of CaCl₂), and distilled water,wherein percentages refer to the mass percentages of the liquidpolysaccharide-producing culture medium) for a 24 hours-culture under30° C. to obtain the seed liquid. It is cultured for 24 hours under 30°C. to obtain the inocula. The inocula is transferred into fresh liquidpolysaccharide-producing culture medium at a ratio of 2% (v/v). It iscultured for 72 hours under 30° C. to obtain the fermentation broth.

(2) The Preparation of the Crude Product of Extracellular Polysaccharidein the Fermentation Broth

(2.1) The fermentation broth obtained in step (1) is heated under 95° C.for 10 min. Once cooled down to 15° C., the pH value is adjusted to 4.4with food-grade lactic acid or hydrochloric acid. It stands for 3 hours.It is centrifuged for 10 min (14000 g) The supernatant is taken. 4volumes of 80%-ethanol solution of the supernatant is added under gentlestirring and the mixture is left to stand overnight. It is centrifugedfor 10 min (14000 g). The precipitates are collected. The percentagerefers to the mass percentage of the ethanol solution.(2.2) The precipitate obtained in step (2.1) is dissolved in distilledwater whose temperature is 50° C. to reach a final concentration of0.5%. which percentage refers to the mass-volume percentage of thesolution of precipitate. Once cooled down to 25° C. trichloroacetic acidis added to the solution, such that the final percentage oftrichloroacetic acid is 10%. The percentage refers to the mass-volumepercentage of the solution. The solution stands for 16 hours under 4° C.It is centrifuged or filtered to remove precipitates. The supernatant isobtained. The supernatant is dialyzed with a dialysis bag (Spectrumlabs,U.S.) with a molecular weight cut-off of 1000 Da against distilled waterfor 72 hours. The water is changed every 12 hours to obtain the dialyzedaqueous solution.(2.3) The dialyzed aqueous solution obtained in step (2.2) is vacuumfreeze dried directly. It is vacuum freeze dried under the condition of0.160 mBar, −30° C. for 72 hours to obtain the crude product ofextracellular polysaccharide. The absorbance at A490 nm by sulfuricacid-phenol method and A280 nm are determined to determine the purity.

II Purification for the Crude Product of Extracellular Polysaccharide

(1) 100 mg of the crude product of the extracellular polysaccharideobtained in Example 10 is dissolved in the Tris-HCl buffer (0.05 mol/L,pH 7.60) to prepare a solution with the concentration of 5 mg/mL. Thechromatography is performed on the DEAE-Sepharose FF column (D1.6×100cm) (GE Healthcare). The linear gradient elution is conducted with theTris-HCl buffer and in turn the Tris-HCl buffer (0.05 mol/L, pH7.60)which contains 0.2-1.2 mol/L NaCl. The flow rate is 2 mL/min. The liquidis collected in tubes (6 mL per tube). The sulfuric acid-phenol methodis used to determine the absorbance at the wavelength of 490 nm (i.e.the characteristic wavelength for polysaccharide). Based on the tubenumbers corresponding to the absorbance, the plot is drawn to obtain theelution curve A. Thus, a primarily purified component of polysaccharideis obtained, namely, the single peak F1.(2) The aqueous solution obtained from the tube with the numbercorresponding to the single peak (F1) through elution in step (1) iscollected. It is dialyzed with a 1000 Da-dialysis bag in deionized waterfor 72 hours. The vacuum freeze drying (0.160 mBar, −30° C.) isconducted for 72 hours. The component B of extracellular polysaccharideis obtained.(3) The component B of extracellular polysaccharide obtained in step (2)is dissolved in the Tris-HCl buffer to prepare a solution with theconcentration of 5 mg/mL. The chromatography is performed on ionexchange column (wherein the packing is DEAE-Sepharose CL-4B; column:D1.6×100 cm, GE Healthcare). The elution is conducted with the Tris-HClbuffer which contains 0.2-1.2 mol/L NaCl. The flow rate is 2 mL/min. Theliquid is collected in tubes (6 mL per tube). The sulfuric acid-phenolmethod is used for tracking and monitoring. The absorbance is determinedat the wavelength of 490 nm. Based on the tube numbers corresponding tothe absorbance, the plot is drawn to obtain the elution curve B. Thus,the further purified component of polysaccharide is obtained on thebasis of component F1, namely, the single peak F2.(4) The aqueous solution obtained from the tube with the numbercorresponding to the single peak (F2) through chromatography in step (3)is collected. It is dialyzed with a 1000 Da-dialysis bag in deionizedwater for 72 hours. The vacuum freeze drying (0.160 mBar, −30° C.) isconducted for 72 hours to obtain the purified extracellularpolysaccharide.

Example 13 Preparation and Purification for Extracellular Polysaccharideof the Strain BD3526 I Preparation for Polysaccharide of Strain BD3526(1) The Fermentation of Paenibacillus and the Preparation forFermentation Broth

10.0 mg of the freeze-dried powder of Paenibacillus BD3526 is dissolvedwith 1 mL of sterile distilled water (100 mg/0.1 ml, impossible!). Oneloop of the suspension is picked and streaked on the solidpolysaccharide-producing culture medium (the solidpolysaccharide-producing culture medium is consisted of 1.2% of agar,10% of saccharose, 1% of casein tryptone, 5% of yeast extract, 5% ofK₂HPO₄, 0.034% of CaCl₂), and distilled water, wherein the percentagesrefer to the mass percentages with respect to thesolid-polysaccharide-producing culture medium). It is aerobicallycultured for 24 hours at 30° C.

The single colony formed on the solid polysaccharide-producing culturemedium is picked up and transferred into the liquidpolysaccharide-producing culture medium (consisting of 10% ofsaccharose, 1% of casein tryptone, 0.5% of yeast extract, 0.5% ofK₂HPO₄, 0.034% of CaCl₂), and distilled water, wherein the percentagesrefer to the mass percentages with respect to the liquidpolysaccharide-producing culture medium). It is cultured for 24 hoursunder 30° C. to obtain the inocula. The inocula is transferred intofresh liquid polysaccharide-producing culture medium at a ratio of 1%(v/v). It is cultured for 72 hours under 30° C. to obtain thefermentation broth.

(2) The Preparation of the Crude Product of Extracellular Polysaccharidein the Fermentation Broth

(2.1) The fermentation broth obtained in step (1) is heated under 100°C. for 30 min. It is cooled down to 25° C. The pH value is adjusted to4.8 with food-grade lactic acid or hydrochloric acid. It stands for 5hours. It is centrifuged for 10 min (14000 g), The supernatant is taken.80%-ethanol solution with the volume twice as much as that of thesupernatant is added into the centrifuged supernatant mentioned above.It stands overnight. It is centrifuged for 10 min (14000 g). Theprecipitates are collected. The percentage refers to the mass percentageof the ethanol with respect to the ethanol solution.(2.2) The precipitates obtained in step (2.1) is dissolved withdistilled water whose temperature is 80° C. to reach a finalconcentration of 1%. The percentage refers to the mass-volume percentageof the solution of precipitates. When the solution is cooled down to 25°C., the trichloroacetic acid is added, such that the final percentage ofthe trichloroacetic acid is of 6%. The percentage refers to themass-volume percentage of the solution. The solution stands for 16 hoursunder 4° C. It is centrifuged or filtered to remove precipitates. Thesupernatant is obtained. The supernatant is dialyzed using a dialysisbag (spectrumlabs, U.S.) which has a molecular weight cut-off of 1000 Dain distilled water for 72 hours. The water is changed every 12 hours.The dialyzed aqueous solution is obtained.(2.3) The dialyzed aqueous solution obtained in step (2.2) is vacuumfreeze dried directly. It is vacuum freeze dried under the condition of0.160 mBar, −30° C. for 72 hours to obtain the crude product ofextracellular polysaccharide. The absorbance at A490 nm and A280 nm isdetermined to determine the purity.

II Purification for the Crude Product of Extracellular Polysaccharide

(1) 100 mg of the crude product of the extracellular polysaccharideobtained in Example 10 is dissolved in the Tris-HCl buffer (0.05 mol/L,pH7.60) to prepare a solution with the concentration of 20 mg/mL. Thechromatography is performed on the DEAE-Sepharose FF column (D1.6×100cm) (GE Healthcare). The linear gradient elution is conducted with theTris-HCl buffer and in turn the Tris-HCl buffer (0.05 mol/L, pH7.60)which contains 0.2-1.2 mol/L NaCl. The flow rate is 6 mL/min. The liquidis collected in tubes (6 mL per tube). The sulfuric acid-phenol methodis used to determine the absorbance at the wavelength of 490 nm (i.e.the characteristic wavelength for polysaccharide). Based on tube numberscorresponding to the absorbance, the plot is drawn to obtain the elutioncurve A. Thus, the primarily purified component of polysaccharide isobtained, namely, the single peak F1.(2) The aqueous solution obtained from the tube with the numbercorresponding to the single peak (F1) through the elution in step (1) iscollected. It is dialyzed with a 1000 Da-dialysis bag in deionized waterfor 72 hours. The vacuum freeze drying (0.160 mBar, −30° C.) isconducted for 72 hours to obtain the component B of extracellularpolysaccharide.(3) The component B of extracellular polysaccharide obtained in step (2)is dissolved in the Tris-HCl buffer to prepare a solution with theconcentration of 20 mg/mL. The chromatography is performed on the ionexchange column (the packing is DEAE-Sepharose CL-4B; column: D1.6×100cm, GE Healthcare). The elution is conducted using the Tris-HCl bufferwhich contains 0.2-1.2 mol/L NaCl. The flow rate is 6 mL/min. The liquidis collected in tubes (6 mL per tube). The sulfuric acid-phenol methodis used for tracking and monitoring. The absorbance at the wavelength of490 nm is determined. Based on the tube numbers corresponding to theabsorbance, the plot is drawn to obtain the elution curve B. Thus, afurther purified component of polysaccharide is obtained on the basis ofcomponent F1, namely, the single peak F2.(4) The aqueous solution obtained from the tube with the numbercorresponding to the single peak (F2) through chromatography in step (3)is collected. It is dialyzed with a 1000-Da-dialysis bag in deionizedwater for 72 hours. The vacuum freeze drying is conducted under thecondition of 0.160 mBar, −30° C. for 72 hours to obtain the purifiedextracellular polysaccharide.

Example 14: Structural Analysis for Extracellular Polysaccharide (1)Infrared (FI-IR) Analysis

2.0 mg of the purified extracellular polysaccharide obtained in Example11 and KBr are ground and tableted. The infrared multispectral scan (XuGuangtong, Yuan Hongfu, Lu Wanzhen, et al, Development of Modern NearInfrared Spectroscopic Techniques and Its Applications, SPECTROSCOPY ANDSPECTRAL ANALYSIS, 2000, 02: 134-142) (infrared spectrometer: ThermoFisher Scientific) is conducted within the area of 4000-500 cm⁻¹.

The infrared spectrogram is shown in FIG. 7. Detailed data are shown inTable 7.

TABLE 7 data of infrared spectrogram Wave number of Type of absorptionpeak characteristic cm⁻¹ Functional group Vibration mode absorption peak3389 —OH OH stretching vibration 2937 —CH C—H stretching vibrationcharacteristic peak of polysaccharide 2882 —CH C—H stretching vibrationcharacteristic peak of polysaccharide 1635 —OH OH bending vibration 1127C—O—C (on ring) C—O stretching vibration 1014 —OH OH deformationvibration 1060 —OH OH deformation vibration 927 furan ring symmetricalstretching vibration of furan ring 809 —CH C—H deformation vibration offuran ring

(2) Nuclear Magnetic Resonance (NMR) Analysis

10 mg of the purified extracellular polysaccharide obtained in Example11 is dissolved in 1 mL of heavy water (D₂O). The ¹H-NMR and ¹³C-NMRspectrums are determined using JNM-A500 (manufacturered by NipponDenshi). The ¹³C-NMR spectrum is shown in FIG. 8. The specific data of¹H-NMR spectrum are as follows: δ(3.538)-(3.585) ppm is H-6b, δ(3.661)-(3.692) ppm is H-lb; δ (3.748)-(3.793) ppm is H-la; δ (3.894)ppm is H-6a; δ (3.935) ppm is H-5; δ (4.082)-(4.132) ppm is H-4; and δ(4.082)-(4.132) ppm is H-4, δ (4.184)-(4.205) ppm is H-3. In FIG. 8,δ(62.67) is C-1, δ(66.46)-δ(66.25) is C-6; δ(77.38)-δ(77.98) is C-4;δ(79.07) is C-3; δ(83.07)-δ(83.92) is C-5; and δ(79.07) is C-2.Therefore, comparing with the standard spectrum for glycosidic bond offructose, these data indicate that the extracellular polysacchariderefers to the levan jointed with β (2→6) glycosidic bond. Meanwhile, theresults of NMR data also indicate that the purified extracellularpolysaccharide obtained in Example 11 comprises no characteristic peaksof a non-carbohydrate structure such as protein, sulfide and amidogen,namely, it is a pure levan with no impurities (for example, protein)contained.

Example 15: Molecular Weight Distribution of the Purified ExtracellularPolysaccharide

(1) The standard samples with different molecular weights are sampledconsecutively. The retention time TR is recorded. A standard curve isdrawn, wherein the horizontal axis refers to retention time RT and thevertical axis refers to 1 gM, so as to get the regression equationbetween the molecular weight and the retention time RT.(2) The purified extracellular polysaccharide to be tested is dissolvedin ddH₂O to reach a concentration of 10 g/L. Sampling is conducted toobtain TR. The relative molecular weights of the samples are calculatedthrough regression equation.Chromatographic conditions are as follows:Chromatographic instrument: Viscotek TDAmax (waters China)Detector: refractive index detector (waters China)Chromatographic column: GPCmax™ range: 2000-20000000 (waters China)Moving phase: 0.1 mol/L NaNO₃Column temperature: 30° C.; Flow rate: 1 mL/min; Concentration ofsample: 10 mg/mL

The results of molecular weight distribution of the polysaccharidementioned above are shown in FIG. 9. The results indicate that: anabsorption peak appears at the retention time of 9.7 min. Comparing withthe chromatogram of polysaccharide which has a standard molecularweight, the polysaccharide is formed by single polysaccharide. The rangeof average molecular weight of the polysaccharide mentioned above is2500-5000 Da. About 90% of the extracellular polysaccharide moleculeshave a molecular weight within the range of 2500-5000 Da, wherein thepercentage refers to the mole percentage of the extracellularpolysaccharide.

Example 16: Analysis on Monosaccharide Composition of the PurifiedExtracellular Polysaccharide

10 mg of the purified extracellular polysaccharide is dissolved in 2 mLof the 0.05 mol/L H₂SO₄ solution. It is hydrolyzed for 2 hours under 80°C. to obtain the hydrolysate A. The hydrolysate A is diluted 100 timesto obtain the hydrolysate B. The monosaccharide composition ofhydrolysate B is analyzed by the high performance ion chromatography(HPAEC-PAD) (Suo Hui, The Primary Structure of Garlic Fructan (D). JinanUniversity, Guangdong, 2010) The determination conditions for ionchromatography are: chromatographic column: (Carbopac, China): Carb 1,4×250 mm; flow rate: 1.0 mL/min; sample size: 20 μL; detector: pulsedamperometric detector (Carbopac, China), gold electrode; temperature fordetermination: 30° C. The isocratic elution is conducted with 15 mM NaOHsolution. The experimental results are shown in FIG. 10. The resultsshow that a peak of about 4200 nA is produced by fructose at 14:36 min.The sample of the purified extracellular polysaccharide produces a peakat 14:54 min. The peak appears at the same time as that of fructose.This indicates that the purified extracellular polysaccharide isconsisting of single fructose. The results of examples 14 to 16 showthat the constitutional repeating unit of extracellular polysaccharideis fructose. the fructose is jointed by means of→2,6-fructose→2,6-fructose, the extracellular polysaccharide refers tolevan and the structural formula of the extracellular polysaccharide isshown as Formula (1),

wherein, n=15˜30, and the extracellular polysaccharide has an appearanceof pure white filament or powder.

Example 17 the Proliferation of B. infantis Promoted by ExtracellularPolysaccharide In Vitro

(1) The faeces of 30 infants (with the age range from 6 to 24 months,excluding the those who have intestinal diseases or taking antibioticdrugs recently) is collected. The standard culture solution of fecalflora is prepared in accordance with the method of Minekus et al (Modelsof the gastrointestinal tract to study microbial interactions OriginalResearch Article Biology of Growing Animals, 2005, 2:142-154).(2) Certain amount of standard ileum efflux culture medium (STEM, with0.047 g/L pectin, 0.047 g/L xyloglucan, 0.047 g/L arabinogalactan, 0.047g/L amylose, 0.392 g/L starch, 24.0 g/L casein, 17.0 g/L Tween 80, 24.0g/L bacto peptone, 0.4 g/L oxgall, and 0.2 g/L cysteine) is added into a96-well microtitration plate. Then, the 0.1% of the standard culturesolution of fecal flora is added. The percentage refers to the volumepercentage of the standard ileum efflux culture medium. Then, 0.4% ofthe purified extracellular polysaccharide (“BD3526 extracellularpolysaccharide” for short) obtained in Example 11 and 0.4% of thecommercialized fructooligosaccharide (FOS for short) are added intodifferent wells among the 96 wells respectively. The final volume ofliquid in each well is 1.5 mL. Meanwhile, the blank control is arranged.They are anaerobically cultured for 8 hours under 37° C. The percentagerefers to the mass-volume percentage of the final volume of the liquidin a well of the 96-well microtitration plate.(3) Once the culture is completed, the fermentation broth is mixed with250 μL of lysis buffer, 250 μL of zirconium bead liquid (0.1 mm) and 200μL of phenol solution. Then they are homogenized on the bead beater (BIOSPEC Inc. U.S.) for twice (2 minutes for each time). The DNA isextracted with DNA Kit (TIANGEN BIOTECH CO., LTD). Then, PCR-intestinalmicroarray detection method (SALAZAR N, GUEIMONDE M, HERNANDEZ-BARRANCOA M, et al. Exopolysaccharides produced by intestinal Bifidobacteriumstrains act as fermentable substrates for human intestinal bacteria.Applied and environmental microbiology, 2008, 74(15): 4737-45) is usedto analyze the number of floras in samples with respect to that of theblank control (standard solution of fecal flora cultured without addingpolysaccharide). The experimental results are shown in FIG. 11 and Table8 (wherein, the log (ORF/mL) value of all floras in the blank control isdefined as 0). As shown in FIG. 11, comparing with the blank controlgroup, Paenibacillus BD3526 EPS (e.g, FOD) can also promote theproliferation of the bifidobacteria in infants' fecal flora, especiallythe proliferation of B. breve. As shown in Table 8, comparing with theblank control, the number of bifidobacteria in floras is increased forboth samples with added FOS and BD3526 extracellular polysaccharide.Especially for B. breve, the increment of number (log (ORF/mL) value) ofthe sample with added BD3526 extracellular polysaccharide is 0.81, whichalmost reaches that (1.18) of the sample with added FOS. For B. longum,the increment of number (log (ORF/mL) value) of the sample with addedBD3526 extracellular polysaccharide is 0.36, which approximates that(0.37) of the sample with added FOS added.

TABLE 8 the number of strains in the flora cultured for 8 hours Theincreased log (ORF/mL) value of the flora in samples with respect to theblank control Treatment for All All culturing bacteria bifidobacteria B.breve B. infantis B. Longum Blank control 0 0 0 0 0 group BD3526 0.4480.74 0.81 0.017 0.36 extracellular polysaccharide FOS 0.12 1.07 1.18−0.121 0.37

Example 18 Effect of Adjusting the Composition of Intestinal Flora forAdults In Vitro

(1) The faeces of a healthy adult (male or female, with the age rangefrom 23 to 25, excluding those who have intestinal diseases or takingantibiotic drugs recently). They are dissolved in pH7.3-phosphate buffer(8 g/L-NaCl, 0.2 g/L-KCl, 1.15 g/L-NaHPO₄, and 0.2 g/L-KH₂PO₄) and mixeduniformly. It is added into the culture system at a volume ratio of1:10. Then, 0.4% of the purified extracellular polysaccharide (BD3526EPSfor short) obtained in Embodiment 11 and 0.4% of the commercializedfructooligosaccharide (FOS for short) are added into the culture systemrespectively. Meanwhile, the blank control is arranged, They areanaerobically cultured for 24 hours under 37° C. The percentages referto the mass-volume percentages of the culture system.(2) Once the culture is completed, the phenol-chloroform method(ERCOLINI D, HILL P J, DODD C E. Bacterial community structure andlocation in Stilton cheese. Applied and environmental microbiology,2003, 69(6): 3540-8) is used to extract the DNA of the cultures andfecal flora., Then PCR-DGGE (denaturing gel gradient electrophoresis)method is used (Analysis of inherited and acquired mutations using PCRand denaturing gradient gel electrophoresis (DGGE) MutationResearch/Environmental Mutagenesis and Related Subjects, Volume 252,Issue 2, 1991, 175-176. A.-L. Borresen, E. Hovig, B. Smith-S0rensen, S.Lystad, A. Bragger) to analyze the composition of the flora of thesamples. That is, the universal primer 357F (as shown in SEQ ID NO. 4)(5′-TACGGGAGGCAGCAG-3′), 518R (as shown in SEQ ID NO. 5)(5′-ATTACCGCGGCTGCTGG-3′) of 16S rDNA for the bacteria, and GC-clamp (asshown in SEQ ID NO. 6)(5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCC-3′) are used. The PCRamplification system is 25 μL (8.5 μL of sterile double distilled water,10_, of each of primer 518R, 10_, of GC-357F, 2 μL of template, and 12.5μL of 2× Plus-Mix). The amplification protocol is: 5 min under 94° C.;then 30 s under 94° C., 30 s under 56° C., 40 s under 68° C., 35 cycles;at last, 10 min under 72° C. for extension. The amplification productsare added onto the gel with a polyacrylamide concentration of 8% (wt)and a denaturing gradient range from 40% (wt) to 60% (wt).Electrophoresis is conducted under 60° C. using the voltage of 75V with1×TAE as the electrophoresis buffer. Once the electrophoresis iscompleted, stain the gel with ethidium bromide. Photographs are takenwith the gel imaging system. The results are shown in FIG. 12. Thestripes pointed by arrows in FIG. 12 are significantly brighter thanthose of the FOS group and the blank control group This indicates thatBD3526EPS can promote the proliferation of bacteria of this kind inhuman intestinal flora, and has the ability to adjust the composition ofhuman intestinal flora.

Although descriptions above on embodiments of the present invention aregiven, those skilled in the art shall understand that all these are justexamples of illustration and various changes or modifications may beexecuted in these embodiments without departing from the principle andessence of the present invention. Therefore, the protection scope of thepresent invention shall be limited by the Claims attached.

1. A Paenibacillus sp., wherein a deposit number of the Paenibacillussp. is CGMCC No. 8333, and G+C content of the Paenibacillus sp. is 47.48mol %.
 2. The Paenibacillus sp. of claim 1, wherein a similarity of 16SrRNA between the Paenibacillus sp and a type strain Paenibacillushunanensis FeL05^(T) (ACCC 10718^(T)=CGMCC 1.8907¹=DSM 22170¹) is 96.6%.3. The Paenibacillus sp. of claim 1, wherein main cellular fatty acidsof the Paenibacillus sp. comprise anteiso saturated fatty acid C_(15:0),anteiso heptadecenoic saturated fatty acid, and hexadecanoyl saturatedfatty acid, a content ratio of the anteiso saturated fatty acid C15:0 is59.02%, a content ratio of the anteiso heptadecenoic saturated fattyacid is 11.09%, and a content ratio of the hexadecanoyl saturated fattyacid is 7.66%.
 4. The Paenibacillus sp. of claim 1, wherein DNA homologybetween the Paenibacillus sp. and a type strain Paenibacillus hunanensisFeL05^(T) is 39.8242.10%.
 5. The Paenibacillus sp. of claim 1, whereinDNA homology between the Paenibacillus sp. and a type strainPaenibacillus polymyxa ATCC 842^(T) (=CGMCC 1.4261^(T)=DSM 36^(T)=KCTC3858^(T)) of Paenibacillus is 41.62-48.60%.
 6. A method for culturingPaenibacillus, wherein a deposit number of the Paenibacillus is CGMCCNo. 8333, and G+C content of the Paenibacillus sp. is 47.48 mol %,wherein the method comprises following steps: inoculating thePaenibacillus onto a culture medium; culturing at a temperature of15-40° C. with a pH value of 5.5-8.5.
 7. The method of claim 6, whereinthe temperature for the culturing is 30° C.
 8. The method of claim 6,wherein the pH value for the culturing is 6.0.
 9. The method of claim 7,wherein the pH value for the culturing is 6.0.
 10. The method of claim6, wherein the culture medium for the culturing further comprises lessthan 10% of NaCl by a mass percentage.
 11. The method of claim 7,wherein the culture medium for the culturing further comprises less than10% of NaCl by a mass percentage.
 12. The method of claim 8, wherein theculture medium for the culturing further comprises less than 10% of NaClby a mass percentage.
 13. The method of claim 6, wherein the culturingis shaking culturing.
 14. The method of claim 7, wherein the culturingis shaking culturing.
 15. The method of claim 8, wherein the culturingis shaking culturing.
 16. The method of claim 9, wherein the culturingis shaking culturing.
 17. The method of claim 6, wherein an inoculumdose of the culturing is 2% by a volume percentage.
 18. The method ofclaim 6, wherein the culture medium is TYC culture medium.
 19. Themethod of claim 6, wherein the culturing is conducted under aerobicconditions.
 20. The method of claim 6, wherein the culture medium forthe culturing further comprises less than 10% of NaCl by a masspercentage.